SITE-DIRECTED MUTAGENESIS OF HUMAN THIOREDOXIN - IDENTIFICATION OF CYSTEINE-74 AS CRITICAL TO ITS FUNCTION IN THE EARLY-PREGNANCY FACTOR SYSTEM

Thioredoxin has been identified as a key component of the ''early preg nancy factor'' system, a system of components present in pregnancy ser a which expresses a lymphocyte modifying activity in an assay known as the rosette inhibition assay. Although thioredoxin alone is inactive, addition of thioredoxin to lymphocytes in combination with nonpregnan cy sera or platelet-activating factor results in a positive response. We have changed several amino acids of human thioredoxin by site-direc ted mutagenesis to investigate the residues required for this cooperat ive function. Conversion of the two active site residues (cysteines 32 and 35) to serines results in a protein devoid of classical redox act ivity; however, this protein retained its ability to cooperate with no n-pregnancy sera or platelet-activating factor in the rosette inhibiti on assay. Vertebrate thioredoxins contain an additional conserved pair of cysteine residues in the C-terminal portion of the protein. Changi ng both to serines resulted in no change in redox activity but complet ely abolished function in the rosette inhibition assay. Further study revealed this function was solely dependent on cysteine 74 as conversi on of only cysteine 74 to serine abolished function, whereas replaceme nt of only cysteine 70 with serine had no effect. The nonfunctional mu tants counteracted the action of pregnancy serum in the assay stongly supporting the hypothesis that thioredoxin is an intergral part of the early pregnancy factor system with residue cysteine 74 having an impo rtant role.